Many patients with Congenital Disorders of Glycosylation (CDG) do not have mutations in the 40 known defective genes. We will identify 7 new CDGs in patients and functionally confirm them in their cells and model some defects in zebrafish. Simple mannose therapy is ongoing in one patient and we will test cells from other patients with the same defect or defects in other mannose-requiring steps as a potential treatment. We will identify mannose-selective plasma membrane transporters and Golgi-located UDP-Galactose transporters. Candidate defective genes will be confirmed with newly developed cellular biomarkers to monitor patients' hypo-glycosylation and correction with cDNA complementation or mannose therapy. Previous work using two zebrafish models of CDG show they mimic glycosylation-deficient phenotypes and one responds to mannose therapy. Our group of patients has Sanger-confirmed mutations in two oligosaccharyl-transferase (OST) complex genes, two in the OST-associated TRAP complex genes, in a putative mannose transporter, a known UDP-Gal transporter, and a putative dolichol metabolism gene. A surprisingly high number of CDG cases have mutations in a known pathological gene, ALG1. These patients make an unanticipated biomarker glycan. Strikingly, mannose therapy reduces the amount of that marker glycan in patient cells or serum and appears to improve one patient's neurological pathology. Other patients may benefit from non-toxic mannose therapy, presumably delivered through mannose transporter(s). In AIM1 we will verify these new Congenital Disorders of Glycosylation. In AIM 2, selected disorders will be modeled in zebrafish morphants for phenotypic and biochemical analysis. AIM 3 will identify human Mannose- preferential transporter(s) and novel Golgi UDP-Gal Transporters. Mannose and galactose therapies may be used in the zebrafish models and possibly in these patients. The dramatic appearance of a new ALG1-specific serum biomarker focuses AIM 4 on the basis of non-natural glycan and identifying patients who might respond to mannose therapy. Evidence suggests that patients with 10 other CDG defects may also respond to mannose therapy. This emphasizes the urgency of trying a simple therapy on other patients. Both CDG patients and basic science will benefit when this project is completed.